Morphological changes in the skin of Rana pipiens in response to metabolic acidosis

The skin of Rana pipiens excretes H+ and this excretion is increased by metabolic acidosis. The mitochondria-rich (MR) cells of the skin have been found to mediate this H+ transport. The purpose of this study was to determine if there is a change in the MR cells of the skin during metabolic acidosis and if the isolated split epithelia of frog skin maintains its capacity to excrete H+. Metabolic acidosis was induced by injecting 120 mM NH4Cl (0.025 ml/g body wt) into the dorsal lymph sac three times a day for 2 days. The frogs were sacrificed and collagenase-split skins from the abdomen of normal and metabolic acidotic frogs were mounted between 2-ml chambers. H+ fluxes into both the mucosal and serosal media were measured and reported in units of (nmol) (cm2)-1 (min)-1. An increase in H+ flux was seen on both the mucosal and serosal sides of the acidotic split skins. The isolated epithelia were fixed, postosmicated, and dehydrated in the chamber. They were then embedded in Spurr's resin and 1-micron sections were cut and stained with Paragon multiple stain. Coded slides were used to count various cell types. Sections were randomly selected and approximately 40,000 cells were counted. Four basic cell types were noted and confirmed by TEM photomicrographs; basal (B) cells, granular (G) cells, keratinized cells, and MR cells. The ratio of G + B cells:MR cells in the normal skins was 1.0:0.021. The ratio in acidotic skins was 1.0:0.34. The average percentage of cell population of MR cells in the normal skins was 2.08 + 0.18 and in acidotic skins 3.20 + 0.36 (P less than 0.005). We conclude that the split skin maintains the capacity to acidify the mucosal fluid. Additionally, during metabolic acidosis there is an increased number of MR cells in the skin and this increase may be an adaptive mechanism of the skin to excrete excess H+ during acidosis.     

Histological changes in the skin of Rana pipiens produced by metabolic alkalosis

The frog skin has been shown to excrete various electrolytes, the rates can be altered by varying metabolic conditions. The present study was performed to determine if metabolic alkalosis results in histological changes in the skin that are characteristic of this state. Rana pipiens were loaded with NaHCO3 and skin biopsies obtained (I). These biopsies were compared with biopsies from either control, unloaded frogs (II), or from NaCl loaded (III) frogs. In blind studies of microscopic sections, 13 of 15 biopsies of a mixture of I and II were correctly diagnosed, and similarly, 18 of 20 of I and III were correctly diagnosed (P = 0.0037, and 0.0002, respectively). The changes due to NaHCO3 treatment included; (1) an abundance of large euchromatin cells on or near the surface; (2) changes in the basal cell layer with elongation and rotation of the nuclei; (3) lighter cells in the spinosal layer; and, (4) sometimes the skin became thicker. We conclude that metabolic alkalosis results in characteristic histological changes in the skin, and that this is probably related to the ability of the skin to excrete bicarbonate.     

The role of the skin of Rana pipiens in sulfate excretion

• 1.1. The intact whole skin of Rana pipiens excretes sulfate and this excretion is increased by sulfate loading of the animal,
• 2.2. Ninety-five percent of the excretion of sulfate seen following sulfate loading is via the skin, while a small amount is handled by the urinary system.

     

Chloride conductance in amphibian skin: regulatory control in the skin of Rana pipiens

Chloride conductance across the isolated skin of Rana pipiens shows a voltage-activated component (G(Cl)(V)) which requires the presence of mucosal Cl. G(Cl)(V) is normally low or dormant. It is stimulated by elevated intracellular cAMP, irrespective whether originating from application of ss-adrenergic agonists (isoproterenol), stimulators of the adenylyl-cyclase (forskolin), inhibitors of the phosphodiesterases (isobutyl-methyl-xanthine) or membrane-permeable cAMP analogues (CPT-cAMP). Baseline G(Cl) under inactivating conditions increases also with cAMP dose-dependently. The data indicate that cAMP is a central regulator of the passive, conductive chloride transport across amphibian skin.     

Increased Na+ excretion by the skin of Rana pipiens after NaCl loading

In vivo the frog skin excretes sodium and the sodium excretion is increased in response to a NaCl load. The sodium excretion can be demonstrated in vitro, and the rate of excretion is greater in skin from NaCl-loaded animals than from control, non-loaded animals. Unidirectional 22Na flux experiments on paired frog skins, as well as 22Na and 24Na bidirectional flux experiments measured in vitro, confirm the above finding that net sodium excretion occurs in response to the NaCl load.     

The effects of changes of the tonicity of the bathing fluid upon the tension generated by atrial trabeculae isolated from the heart of the frog, Rana pipiens.

Raising the tonicity of the fluid bathing frog atrial trabeculae has three effects: an initial sustained relaxation, which depends on muscle length and probably originates from structures other than the contractile apparatus; an increase in contractility, which takes the form of a transient contracture if the muscle has previously undergone a high-potassium or a low-sodium contracture, and a further rise in contractility on return to isotonic fluid (off response). The hypertonic contractures, in high-potassium or sodium-free fluids, are antagonized by local anaesthetics and in Na-free media they are unaffected by removal of extracellular coat, whereas the 'off responses' are insensitive to both experimental manoeuvres. Hypotonic fluids applied in Na-free solutions evoke a phasic and a tonic contracture, neither of which are sensitive to local anaesthetics. The tonic response is reduced by lowering the [Ca]o, and occurs at tonicities where the permeability of the cell membrane is likely to have increased. The phasic part of the hypotonic contracture resembles the 'off response' which follows exposure to hypertonic solution. The effects of hypertonic fluids and of caffeine on frog heart are alike, and are also similar to the responses induced by the same experimental manoeuvres in skeletal muscle. The results can be interpreted by assuming that the intracellular relaxing system in frog heart is sensitive to changes in tonicity, and could be functionally divided.     

Speciation in the Rana pipiens Complex

The long held view that leopard frogs (Rana pipiens complex)are a single widely distributed species is not correct. Fivesibling species are currently recognizable. These findings haveimportant implications on the use of leopard frogs in experimentalresearch.     

Cytodifferentiation in the Rana pipiens oocyte

Summary In early diplotene frog oocytes incubated to illustrate thiamine pyrophosphatase (TPPase) activity, reaction product is uniformly distributed within the compartments of the endoplasmic reticulum and nuclear envelope as well as within the saccules and small vesicles comprising the dictyosomes. With continued oocyte development the reaction product becomes concentrated in localized regions of the dictyosome saccules. Eventually, the enzyme is no longer apparent within the endoplasmic reticulum, but is concentrated in the cisternae of the inner dictyosome saccules. The variations noted suggest that the enzyme is synthesized early in diplotene by the endoplasmic reticulum and is subsequently transported to the Golgi apparatus where it is consistently observed at later developmental stages. TPPase activity is also present in the Golgi apparatus of follicle and theca cells as well as in ovarian epithelial cells. The enzyme is also detected in micropinocytotic vesicles contained within the cells comprising the follicle envelope and in intercellular spaces of the follicle. Horseradish peroxidase injected into the coelomic cavity is transported via micropinocytotic vesicles into and through the cells comprising the follicle envelope and in intercellular spaces. The exogenous protein is not found even after a prolonged time period in early diplotene oocytes. The protein is, however, present in large spherical and tubular vesicles in the cortex of vitellogenic oocytes approximately 500 microns in diameter. The possible functional role of the enzyme TPPase during oogenesis is discussed.This investigation was supported by a research grant from the National Science Foundation (GB-8736).     

Circulation of marker substances in the cerebrospinal fluid of an amphibian,Rana pipiens

Summary Solutions of fluorescein-labelled dextran or Evans blue-albumin were infused into the lateral cerebral ventricle of Rana pipiens. The subsequent distribution in the cerebrospinal fluid (CSF) was investigated between 2 and 24 h after infusion by freezing and examination of the cut blocks of the head and vertebral column of the stage of a freezing microtome. These marker substances move out of the ventricles into the subarachnoid space at the caudal end of the fourth ventricle and spread rapidly along the subarachnoid space of the spinal cord. The spreading of marker substances is slower into the brain subarachnoid space. When the marker is infused into the subarachnoid space of the forebrain, it becomes distributed throughout the subarachnoid space of the brain and spinal cord but not in the ventricles.Partial clearance of markers from the ventricles takes place within 5 h and total clearance within 8 h. Clearance from the brain and cord subarachnoid space is somewhat slower and can only be detected in experiments lasting 10 h or more. Absorption of the markers from the CSF occurs via the intervertebral foramina of the spinal cord. Fluorescence microscopy of sections of the cord show that the fluorescence leaves the subarachnoid space at the point where the spinal nerves traverse the arachnoid membrane.     

Carbonic anhydrase activity in Rana pipiens skin: biochemical and histochemical analysis

Summary Carbonic anhydrase activity was demonstrated biochemically and localized histochemically in epidermis of Rana pipiens. The activity observed throughout the lower epidermis was diminished or abolished by periods of fixation which did not affect the activity in upper epidermis. In the latter region, the enzyme was demonstrated only in certain cells with a flask shape known to have numerous mitochondria and a system of convoluted apical membranes. Such findings raise the possibility that these distinctive cells play a role in transport functions of the isolated frog skin suppressed by carbonic anhydrase inhibitors.This study was supported in part by U.S. Public Health Service Grant GM-00568.Student at School of Medicine, Rutgers University.     

Metamorphic changes in localization of sugars in skin of the leopard frog,Rana pipiens

A lectin histochemical study was carried out to determine the distribution of specific sugars in glycoconjugates within an important osmoregulatory organ, amphibian skin. Paraffin sections were made of Rana pipiens skin from dorsal and ventral regions of aquatic larvae in representative developmental stages as well as from several body regions of semiaquatic adult frogs. Sections were incubated with horseradish peroxidase (HRP)‐conjugated lectins, which bind to specific terminal sugar residues of glycoconjugates. Such sites were visualized by DAB‐H₂O₂. The following HRP‐lectins were used: UEA‐1 for α‐L ‐fucose, SBA for N‐acetyl‐D ‐galactosamine, WGA for N‐acetyl‐β‐D ‐glucosamine, PNA for β‐galactose, and Con A for α‐mannose. We found that lectin binding patterns in larvae change during metamorphic climax as the skin undergoes extensive histological remodeling; this results in adult skin with staining patterns that are specific for each lectin and are similar in all body regions. Such findings in R. pipiens provide additional insight into the localization of molecules involved in osmoregulation in amphibian skin. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Immunohistochemical localization of thyrotropin-releasing hormone (TRH) in skin of Rana pipiens

Summary Using immunofluorescent techniques thyrotropin releasing hormone (TRH) is demonstrated in skin of Rana pipiens and R. catesbeiana. The immunofluorescent-TRH is localized in all cell layers of the epidermis and in the epithelium lining the various cutaneous glands, but not in the dermal layer.We wish to thank Dr. Ronald DeLellis and Ms. Mary Blount for their expert advice and guidance in the immunohistochemical techniques.This investigation was supported by NIH National Research Service Award # 1F32 AMO6018-01 from the NIAMDD to Janice L. Bolaffi and NIH Grant AM 21863 to Ivor M.D. Jackson.     

Continuity between the ventricular and subarachnoid cerebrospinal fluid in an amphibian,Rana pipiens

Summary Continuity between the ventricular and subarachnoid cerebrospinal fluid has been investigated in Rana pipiens. The structure of the posterior tela, a deficient membrane situated at the extreme caudal end of the roof of the fourth ventricle, has been studied using whole membrane mounts and by light microscopy of resin embedded tissue. The ependymal component consists of columnar and rounded cells which form a regular syncytium enclosing round and oval fenestrations. Small fenestrations are covered on the subarachnoid side by elongated pial cells and thus do not give total continuity between the fourth ventricle and the subarachnoid space. Large fenestrations, on the other hand, are accompanied by equivalent pial fenestrations giving direct access between the fluid compartments. Towards the caudal end the fenestrations break up and the numbers of ependymal and pial cells decrease, the caudal end itself being characterised by a small remaining clump of ependyma and pia or of pia alone.Flow through the tela has been studied using fluorescein-labelled dextran placed in the intraventricular space. Infusion into the lateral ventricle and subsequent localisation by fluorescence microscopy shows the marker to be in the fourth ventricle, in the fenestrations of the posterior tela and in the subarachnoid space overlying the tela. Infusion of the marker followed by freezing and examination of the cut heads on a freezing microtome, shows fluorescence throughout the ventricular system, in the subarachnoid space adjacent to the posterior tela and also along the dorsal subarachnoid space of the spinal cord.     

Intrinsic pigment cell stimulating activity in the skin of the leopard frog, Rana pipiens.

Consistent with the concept that specific pigment patterns of amphibians might result from the highly localized distribution of stimulators and inhibitors of pigment cell expression in the skin, the spot pattern of the leopard frog, Rana pipiens, was examined through the use of the Xenopus neural tube explant assay system (Fukuzawa and Ide, 1988). Media conditioned with pieces of skin from dorsal black spotted areas promoted melanization of neural crest cells at a significantly higher level than did media conditioned with dorsal interspot skin in the absence of extra tyrosine. All conditioned media contained exceedingly low concentrations of tyrosine. With the addition of supplemental tyrosine, the melanization capacity of conditioned media from the interspot areas was elevated to that of the spotted skin. Control media conditioned with ventral frog skin inhibited melanization, as usual, because of the presumed presence of melanization inhibiting factor (MIF). It is considered that dorsal skin contains a melanization stimulating factor (MSF) which is present in significantly higher levels in spotted skin than in interspot areas and that expression of the particular pigmentary pattern of this leopard frog is regulated by the relative distribution of MIF, MSF, and possibly other intrinsic substances present in the skin.     

Differentiation of the Opercular Integument in Rana pipiens

The opercular skin develops in its own specific fashion differing from other areas of the integument previously studied. It remains typically larval until it is sloughed off.
The opercular integument first deviates from the common skin development pathway at stage XV [1] when the rough endoplasmic reticulum. proliferates to form a large part of the cytoplasm and is accompanied by numerous large mitochondria. The Golgi apparatus becomes very large and many vesicles are found in the cytoplasm.
The mitochondria become large and swollen with atypical structure. The cytoplasm becomes highly vesiculated. Degeneration begins at the dermal edge of the basement lamella and proceeds toward the epidermis, being marked by the disorientation of collagen fibrils.
Epidermal cells become progressively more necrotic having cytoplasm consisting mainly of residual bodies and vesicles: the nucleus is the last cellular structure to undergo autolysis.
The dermal cells are the first to be lost (stages XVIII–XIX). The opercular integument is shed as a sheet of cells at stage XX when the forelimbs emerge. The necrotic zone of the opercular integument appears to stop at the junction of the body skin and the skin of the forelimb.